Na,K-ATPase isoform-selective cardiac glycosides - a potential anti-cancer drug ?

1. Na,K-ATPase isoform-selective cardiac glycosides- a potential anti-cancer drug ? Adriana Katz, Cherniavsky-Lev M., Ainbinder E., Tal D. and Karlish SJD Weizmann Institute of Science, Israel

2. Ion channels and pumps as cancer targets! Over the years, several reports have suggested that cardiac glycosides may have an anticancer utilization. In vitro and ex vivo experiments have revealed that some cardiac glycosides induce potent and selective anticancer effects, which may occur at concentrations commonly found in the plasma of patients treated with these drugs. Na+ /K+ -ATPase could be targeted to combat chemoresistant cancers.

3. Na,K ATPase is a vital protein in all mammalian cells. • Na,K -ATPase is an oligomerictransmembraneprotein,localized to the basolateral plasma membrane in most epithelial cells. • Na,/K-ATPase pumps Na+ and K+ against their physiological gradients. . . • Subunit • targets into the membrane, 3 isoforms: • b1 • b2 • b3 Digoxin Specific inhibitor Extracellular space membrane FXYD accessory protein, regulator Cytoplasm a subunit Catalytic moiety, 4 isoforms: a1, ubiqutous , all tissues a2, skeletal muscle, heart, eyes a3, neurons brain a4, sperms

4. Na,K pump is essential for many physiological processes a1/b1 • Renal function, and regulation of hypertension . • Cardiac contraction • Regulation of intra ocular pressure, IOP. • And many more…. Expression of isoforms in different tissues. a3b1 a1b1 a2/b2 All of the isoforms are expressed in a tissue and functional specific manner. a3/b1 a1/b1 a1/b1 a2/b3 a2/b1

5. Cardiac glycosides (CG) The cardiac glycosides are an important class of naturally occurring drugs whose actions include both beneficial and toxic effects on the heart. Lactone ring Steroid Glycone

6. Withering W (1785). “An account of the foxglove and some of its medical uses: with practical remarks on dropsy and other diseases.” Digitalis purpurea Strophanthusgratus Cardiac glycosides, naturally occurring in plants and animals. Digoxin Ouabain BufobufoBufalin

7. Cardiac glycosides have a long history of therapeutic application. • Plants containing cardiac steroids have been used as poisons and heart drugs • at least since 1500 B.C. • The early understanding of their positive inotropic effects facilitated their use as effective • drugs for the treatment of heart-related pathologies, yet their toxicity remains a serious problem. • More recently, considerable in vitro, in vivo and epidemiological data support novel roles • for CG’s such as inducing apoptosis and inhibit the growth of cancer cell lines. Control +10uM CG +20uM CG Liang‑FeiYe,et al.2013 Oncology letters

8. Proposed mode of action of cardiac glycosides, CG SlingerlandM. et al Na+,K+ ATPase Na+/Ca+2 exchang. Na+/H+exchang. The decrease in intracellular K+ and increase in intracellular Na+ and Ca2+ following inhibition of the Na+/K+-ATPase may induce apoptosis

9. Na,K-ATPase as a versatile signal transducer ? Na+/K+-ATPase may also act as a signal transducer. When intact cells are exposed to digitalis drugs (e.g., ouabain and digoxin) specific inhibitors of this enzyme various cell signaling pathways are activated leading to highly cell-specific down-stream consequences.

10. Proliferation of CG-treated cells. Normal Cancer cells Breast cancer cells are not more sensitive to CG’s cytotoxicity than are normal cells. Clifford RJ, and Kaplan JH 2013 PLOS ONE 8,

11. Purification and stabilization of isoforms of human Na,K-ATPase expressed in Pichiapastoris

12. 3 a2 a1 MW   Isoforms of human Na,K-ATPase expressed in Pichiapastoris Human isomers/1 purified enzymes Functional, stable, detergent-soluble FXYD complex FXYD1 Detergent , C12E8 Phosphatidylserine,SOPS Phosphatidyl choline, PC Cholesterol Enables different combinations of isoforms

13. Digoxigenin 1.2 1 0.8 0.6 0.4 0.2 0 0.001 0.01 0.1 1 10 1.2 1 0.8 0.6 0.4 Digoxin 0.2 0 0.001 0.01 0.1 1 Cardiac glycoside affinity and selectivity for the  isoforms. • Isoform selectivity is determined by the sugar component! • Digoxin, is partially 2-selective CG, while Ouabain shows very low selectivity    Digoxigenin, μM Bound 3H-Ouabain, fraction of control Digoxin, μM

14. Isoform selectivity of Cardiac glycosides

15. Ouabain Na,K-ATPase structure with bound ouabain The residues common to a2 and a3 and different in a1 are all located on the extracellular loops at the entrance to the Ouabain binding site, close to the sugar moiety.

16. Inhibition of cancer cell proliferation at low concentrations is used to assess the therapeutic potential of drug candidates in preclinical studies. In vitro growth inhibitory concentrations at 50% (IC50) in human cancer cells after three days of culture in the presence of the drug of interest

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19. In vitro growth inhibitory concentrations at 50% (IC50) in human cancer cells after two days of culture in presence of GBR, the most effective CG. 1. TK-10 HCT-116 MCF-7 LLC-PK Cell lines: LLC-PK pig kidney, MCF-7 breast cancer , HCT-116 colon cancer, TK-10 renal cancer.

20. Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects GBR Oleandrin Hellebrin Digoxin Hellebrig. Ouabain

21. Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects 1 2 1. Digoxin 2.Hellebrin 3.Ouabain 4.Oleandrin 5.Hellebrigenin 6. Gamabuf. Rhamnoside 3 5 4 6 4 repetitions with 14 different cancer cells. Cell growth is linearly correleted to the affinity of individual CG to the Na,K-ATPaes

22. Viability is linearly correlated with number of active pumps in partially silenced H1299 cells α1-GFP Si-α1-8 Viability at 30 nMOuabain (% of NT) Si-α1-5 Si-α1-3 Relative binding capacity (%of α1-GFP)

23. mdamb231 mdamb231 lncap lncap llcpk1 mcf7 llcpk1 a549 mcf7 a549 hela hela skmel5 skmel5 panc1 panc1 hek hek Viability (%of NT) Viability (%of NT) ovcar3 ovcar3 hff h1299 h1299 hff Bmax (pmoloub*mg protein-1) Total α1 (a.u.) Viability is linearly correlated with number of active pumps in a panel of cells h1299 hff llcpk1 lncap mda-mb-231 a549 skmel5 hela panc1 hek mcf7 ovcar3 A 50 ng Pur. Pr. α1α2 α3 anti α1 anti α2 anti α3 B C

24. Viability is linearly correlated with number of active pumps– CG affect cancer cell viability • only through binding and inhibition of NaK ATPase transport • Although cardiac glycosides can inhibit the proliferation of cancer cells at very low concentrations (nM), they inhibit the proliferation of human nonmalignant • cells at similar concentrations; this strongly suggests that their potential for cancer therapy is low. • More experimental data are needed to further decipher the structure-activity relationship between CG’s and cancer cell cytotosicity.

25. Acknowledgments Steven Karlish Marina Cherniavsky-Lev Elena Ainbinder Daniel Tal YotamNadav Michael Habeck Thank you for your attention.